Capillary electrophoresis is a technique for analyzing and/or purifying a wide variety of biochemical substances or analytes such as proteins, nucleic acids, carbohydrates, hormones and vitamins. In particular, electrophoresis is an extremely efficacious and powerful means for the identification and/or separation of analytes based upon ultra-small volume samples. In general, electrophoresis is a phenomenon that involves the migration of charged particles or analytes through a conducting liquid solution under the influence of an applied voltage.
The basic capillary electrophoretic apparatus consists of a capillary column having the ends thereof positioned in reservoirs containing electrodes. A conducting liquid or buffer solution disposed in the reservoirs and the capillary column comprises the electrophoretic conductive circuit.
An analyte is injected into the appropriate end of the capillary column and a voltage applied across the electrodes. The applied voltage causes the analyte to migrate electrophoretically through the capillary column past a prepositioned on-column detection device to generate an electropherogram, a graphical representation of the analyte.
Electrophoresis may be conducted in "open" or "gel" capillary columns. Open capillary electrophoresis can be conducted either with or without electroosmosis which involves bulk solvent migration under the influence of the applied voltage as a result of the charged condition of the inner wall of the capillary column. Gel capillary electrophoresis, in which the interior channel of the capillary column is filled with a suitable gel, provides the potential for different modes of separation based upon size of the analytes.
In either open or gel capillary electrophoresis, however, the applied voltage is a primary factor affecting the migration of the analyte. Therefore, the term electromigration as used herein encompasses either or both forms of voltage induced analyte movement.
An effective high performance capillary electrophoretic system provides high resolution, high sensitivity, short run times, on-line monitoring or detection of the analyte, and reproducible performance. One practical way of enhancing the performance of a capillary electrophoretic apparatus is by the application of high applied voltages. Another is to utilize shortened capillary columns. Both of these means of enhancing the effectiveness of the electrophoretic apparatus, however, have heretofore been limited due to the Joule heat generated in the capillary column during the electrokinetic separation operation which adversely affects electrophoretic separations.
The applied voltage causes a current flow in the buffer solution of the electrophoretic apparatus. The current flow through the capillary column generates Joule heat or thermal energy in the capillary column. Increasing the applied voltage increases the current flow which increases the amount of Joule heat generated, which is generally an adverse condition since most electrophoresis is optimally conducted at low, constant temperatures. Similarly, shortening the length of the capillary column decreases the capillary column resistance, thereby causing an increase in current flow for a given applied voltage with the concomitant increase in Joule heating.
An optimized capillary electrophoretic apparatus provides statistically reproducible results for equivalent analytes, with minimum band broadening of the output. Preferably, the apparatus is operated at high applied voltages to provide high speed, efficiency and resolution of separations.
Prior art attempts t cool capillary electrophoretic apparatus have typically involved the use of water as the cooling element. In additional to being high capacity devices, requiring two to four liters of water, water-cooled devices suffered a marked degradation in cooling performance, about 20 to 40 percent, at temperatures approaching four degrees centigrade. Another limiting aspect of prior art capillary electrophoretic apparatus was due to the fact that the structural configuration of prior art temperature regulating systems severely hindered temperature control in the detection zone. A lack of temperature control can lead to non-reproducible results in migration rates and separation.
Moreover, reproducible results in signal-to-noise ratio were difficult to achieve in prior art capillary electrophoretic apparatus due to the cumbersome and time consuming effort required to properly align and lock the capillary column with respect to the prepositioned detection device. Improper alignment of or failure to lock the capillary column in a predetermined position leads to the generation of variable noise due to vibration effects, which can lead to poor detection limits.